Rapid Elution of 226Th from a Two-Column 230U/226Th Generator with Diluted and Buffer Solutions

A radionuclide generator of the short-lived alpha emitter 226Th was proposed. An original scheme consisting of two in-series chromatographic columns was developed for rapidly producing a neutral citric-buffered eluate of high purity 226Th. The first column filled with TEVA resin retained the parent 230U, while 226Th was eluted with 7 M HCl solution to be immediately adsorbed on the second column containing DGA resin or UTEVA resin. Having substituted the strongly acidic medium of second column with neutral salt solution, 226Th was desorbed with diluted citric buffer solution. One cycle of generator milking took 5–7 min and produced >90% of 226Th in 1.5 mL of eluate (pH 4.5–5.0) appropriate for direct use in radiopharmaceutical synthesis. The 230U impurity in 226Th eluate was less than 0.01%. The proposed two-column 230U/226Th generator was tested over 2 months including a second loading of 230U additionally accumulated from 230Pa.


Introduction
A new fast-paced branch of nuclear medicine called targeted alpha therapy (TAT) is effective for the treatment of various oncological diseases due to the property of αparticles to release a large amount of energy in a limited area of living tissue (~10 cell diameters). One of the promising radionuclides for TAT is 230 U (T 1/2 = 20.2 d) [1]. The decay of 230 U generates a chain of short-lived products that emit five α-particles with a total energy of 33.5 MeV (Figure 1), resulting in effective cell damage [2]. The short-lived daughter alpha emitter 226 Th (T 1/2 = 30.6 min) is also an attractive radionuclide for using in TAT [3]. In terms of nuclear properties, the 230 U/ 226 Th pair is similar to the well-researched 225 Ac/ 213 Bi generator pair [4].
The expected therapeutic efficacy of 230 U/ 226 Th has not been demonstrated yet; nevertheless, research is underway to find optimal chelating agents that can stably bind in vivo 230 U [5] and 226 Th [6]. Daughter nuclide released from a radiopharmaceutical molecule due to recoil effect can potentially migrate from the target cancer cell, which might cause considerable toxic effects to heathy tissues. If the half-life of a formed nucleus is short, its diffusion is time-limited. Therefore, a major advantage of 226 Th-TAT compared with 230 U-TAT is that the uncontrolled redistribution of a recoiled daughter nuclide (for 230 U, this nuclide is 226 Th itself) is significantly diminished (Figure 1).
The effective and reliable production of 230 U and 226 Th is required in order to accelerate biological studies and implement these radionuclides in TAT. 230 U can be produced directly via nuclear reactions 231 Pa(p,2n) 230 U [7] and 231 Pa(d,3n) 230 U [8]. The initial 231 Pa (T 1/2 = 3.3 × 10 4 y) is a decay product of 235 U, it must be isolated from aged uranium samples. The raw material is hardly accessible, which restricts the method implementation. Another approach uses reactions of thorium nuclei with accelerated protons and deuterons, leading implementation. Another approach uses reactions of thorium nuclei with accelerated protons and deuterons, leading to the formation of the 230 Pa precursor decaying into 230 U with a branching ratio of 7.8%: 232 Th(p,3n) 230 Pa→ 230 U and 232 Th(d,4n) 230 Pa→ 230 U. Scientific organizations worldwide are actively developing the production of 230 U through irradiation of 232 Th with protons [9][10][11][12][13]. This method has proven to be the most effective in terms of product yield compared with the reactions with deuterons [14,15]. Moreover, the maximum of the 232 Th(p,3n) 230 Pa reaction excitation function is around 20 MeV [15,16], which enables up-scaled production of 230 U on accessible commercial cyclotrons. At higher proton energies (>100 MeV), 230 U can be obtained as a byproduct in 225 Ac production [17,18] along with 223 Ra [19]. 230 U from proton-irradiated thorium contains a chemically inseparable impurity of long-lived uranium isotopes. This impurity was evaluated in our previous paper [15] as up to 0.02% 232 U (T1/2 = 68.9 y) and 0.001% 233 U (T1/2 = 1.6 × 10 5 y). Long-lived admixtures make the medical use of 230 U as a source in a radionuclide generator of 226 Th more prospective than direct applications. 226 Th is considered to be an alternative to another promising generator-produced short-lived radionuclide 213 Bi (T1/2 = 45.6 min) [20]. 226 Th presumably provides a greater impact on cancer cells compared with 213 Bi. A rapid cascade of four α-particles initiated by 226 Th decay deposits totally 27.7 MeV, while 213 Bi emits only one α-particle with an energy of 8.4 MeV. 226 Th-radiopharmaceuticals can be effective for therapy of epithelial or easily accessible tumors [21]. Radioimmunoconjugates Nimotuzumab-p-SCN-Bn-DTPA(DOTA) were synthesized in our previous paper and their specificity toward EGFR overexpressing epidermoid carcinoma A431 cells were demonstrated [22].
Due to the relatively short half-life of 226 Th, time economy becomes a major requirement throughout the entire process from obtaining 226 Th to radiopharmaceutical administration. For this reason, the generator system must ensure the rapid and efficient separation of the accumulated thorium radionuclide. Various methods of liquid-liquid extraction [23,24], extraction chromatography [11,[25][26][27], and ion exchange chromatography [28][29][30] have been developed for the separation of thorium and uranium. For example, the methods employing extraction chromatographic sorbents TEVA TM resin Scientific organizations worldwide are actively developing the production of 230 U through irradiation of 232 Th with protons [9][10][11][12][13]. This method has proven to be the most effective in terms of product yield compared with the reactions with deuterons [14,15]. Moreover, the maximum of the 232 Th(p,3n) 230 Pa reaction excitation function is around 20 MeV [15,16], which enables up-scaled production of 230 U on accessible commercial cyclotrons. At higher proton energies (>100 MeV), 230 U can be obtained as a byproduct in 225 Ac production [17,18] along with 223 Ra [19]. 230 U from proton-irradiated thorium contains a chemically inseparable impurity of long-lived uranium isotopes. This impurity was evaluated in our previous paper [15] as up to 0.02% 232 U (T 1/2 = 68.9 y) and 0.001% 233 U (T 1/2 = 1.6 × 10 5 y). Long-lived admixtures make the medical use of 230 U as a source in a radionuclide generator of 226 Th more prospective than direct applications. 226 Th is considered to be an alternative to another promising generator-produced short-lived radionuclide 213 Bi (T 1/2 = 45.6 min) [20]. 226 Th presumably provides a greater impact on cancer cells compared with 213 Bi. A rapid cascade of four α-particles initiated by 226 Th decay deposits totally 27.7 MeV, while 213 Bi emits only one α-particle with an energy of 8.4 MeV. 226 Th-radiopharmaceuticals can be effective for therapy of epithelial or easily accessible tumors [21]. Radioimmunoconjugates Nimotuzumab-p-SCN-Bn-DTPA(DOTA) were synthesized in our previous paper and their specificity toward EGFR overexpressing epidermoid carcinoma A431 cells were demonstrated [22].
Due to the relatively short half-life of 226 Th, time economy becomes a major requirement throughout the entire process from obtaining 226 Th to radiopharmaceutical administration. For this reason, the generator system must ensure the rapid and efficient separation of the accumulated thorium radionuclide. Various methods of liquid-liquid extraction [23,24], extraction chromatography [11,[25][26][27], and ion exchange chromatography [28][29][30] have been developed for the separation of thorium and uranium. For example, the methods employing extraction chromatographic sorbents TEVA TM resin and UTEVA/TRU TM resin were recommended for the selective isolation and determination of Molecules 2023, 28, 3548 3 of 17 U, Th, and a number of other radionuclides in water (sample volume up to 1 L) [31,32]. The chromatographic methods are more appropriate for a 230 U/ 226 Th generator since they usually provide 226 Th in a small volume of eluate containing a reduced amount of long-lived impurities.
Effective separation of U(VI) and Th(IV) can be achieved on sorbents displaying anionexchange properties in strong hydrochloric acid solutions. As it can be seen in Figure 2, U(VI) exhibits high affinity to a strong base anion-exchange resin Dowex 1 (or AG 1) and to an extraction chromatographic resin TEVA at c(HCl) > 6 M, whereas Th(IV) is not retained. Both resins were tested as sorbents for a 230 U/ 226 Th «direct» generator [22]. TEVA resin proved to be more preferable, it provided high 226 Th yield in less volume of eluate (1-2 mL). Furthermore, the maximal mass distribution ratio D m of U(VI) adsorbed on TEVA resin is located around 7 M HCl, whereas the largest adsorption of U(VI) on AG 1 corresponds to HCl concentration greater than 9 M (Figure 2). The high acidity of the final 226 Th eluate was found to be the main disadvantage assuming the extra-time needed to convert the eluate into neutral solution prior to labeling. and UTEVA/TRU TM resin were recommended for the selective isolation and determination of U, Th, and a number of other radionuclides in water (sample volume up to 1 L) [31,32]. The chromatographic methods are more appropriate for a 230 U/ 226 Th generator since they usually provide 226 Th in a small volume of eluate containing a reduced amount of longlived impurities. Effective separation of U(VI) and Th(IV) can be achieved on sorbents displaying anion-exchange properties in strong hydrochloric acid solutions. As it can be seen in Figure  2, U(VI) exhibits high affinity to a strong base anion-exchange resin Dowex 1 (or AG 1) and to an extraction chromatographic resin TEVA at c(HCl) > 6 M, whereas Th(IV) is not retained. Both resins were tested as sorbents for a 230 U/ 226 Th «direct» generator [22]. TEVA resin proved to be more preferable, it provided high 226 Th yield in less volume of eluate (1-2 mL). Furthermore, the maximal mass distribution ratio Dm of U(VI) adsorbed on TEVA resin is located around 7 M HCl, whereas the largest adsorption of U(VI) on AG 1 corresponds to HCl concentration greater than 9 M (Figure 2). The high acidity of the final 226 Th eluate was found to be the main disadvantage assuming the extra-time needed to convert the eluate into neutral solution prior to labeling. Another approach is based on a reverse scheme of a 230 U/ 226 Th generator, i.e., the parent 230 U is not fixed on the column filled with sorbent while the daughter 226 Th remains adsorbed. An extraction chromatographic DGA resin containing a diglycolamide derivative was reported to be appropriate for obtaining 226 Th in citric buffer solution that was amenable to direct labeling with minimal losses of time [13]. However, the 226 Th eluate recovered from the reported reverse 230 U/ 226 Th generator contained at least 0.2% of 230 U [13], which was unacceptable for clinical trials to date.
In the presented article, we investigated different schemes of two-column 230 U/ 226 Th generators pursuing two goals: (i) Obtaining 226 Th of high purity in a solution amenable to further labeling; (ii) reducing the time of 226 Th production. The first column served for fixing the parent 230 U and elution of 226 Th. The second column was intended to adsorb 226 Th from a strongly acidic solution, and then to desorb it with diluted or neutral solution. This concept was proposed earlier and tested for a 225 Ac/ 213 Bi generator [34][35][36]. In the first column, 225 Ac formed an extremely strong complex with bis-  Another approach is based on a reverse scheme of a 230 U/ 226 Th generator, i.e., the parent 230 U is not fixed on the column filled with sorbent while the daughter 226 Th remains adsorbed. An extraction chromatographic DGA resin containing a diglycolamide derivative was reported to be appropriate for obtaining 226 Th in citric buffer solution that was amenable to direct labeling with minimal losses of time [13]. However, the 226 Th eluate recovered from the reported reverse 230 U/ 226 Th generator contained at least 0.2% of 230 U [13], which was unacceptable for clinical trials to date.
In the presented article, we investigated different schemes of two-column 230 U/ 226 Th generators pursuing two goals: (i) Obtaining 226 Th of high purity in a solution amenable to further labeling; (ii) reducing the time of 226 Th production. The first column served for fixing the parent 230 U and elution of 226 Th. The second column was intended to adsorb 226 Th from a strongly acidic solution, and then to desorb it with diluted or neutral solution. This concept was proposed earlier and tested for a 225 Ac/ 213 Bi generator [34][35][36]. In the first column, 225 Ac formed an extremely strong complex with bis-(2ethylhexyl)methanediphosphonic acid (H2DEH[MDP]) immobilized on a silica support (Ac resin). Products of the 225 Ac decay, 221 Fr, and 213 Bi were eluted with 1 M HCl and concentrated on the second column filled with the ion exchanger AG-MP 50 from 0.2 M HCl. Then, 213 Bi was eluted from the second column with 0.1 M HI. The high efficiency of this approach makes it promising for the 230 U/ 226 Th pair, as well.

Results and Discussion
A two-column 230 U/ 226 Th generator was proposed and investigated for fast 226 Th production in diluted or neutral citric solution. The parent 230 U was adsorbed onto the first column filled with TEVA resin (Triskem Int.). On reaching the transient equilibrium, the daughter 226 Th was separated and eluted with strong HCl solution. The role of the second column was to reduce quickly the acidity of 226 Th solution, i.e., a sorbent for second column was to retain 226 Th from the strong HCl solution and to desorb it into a diluted solution. Three extraction chromatographic resins eligible for this purpose were considered: TRU resin, UTEVA resin, and DGA resin (all Triskem Int.). According to the reported data [37][38][39] obtained in static conditions and shown in Figure 3a, the resins can be arranged in a row with respect to Th(IV) retention from <5 M HCl solution: DGA resin > TRU resin > UTEVA resin

Results and Discussion
A two-column 230 U/ 226 Th generator was proposed and investigated for fast 226 Th production in diluted or neutral citric solution. The parent 230 U was adsorbed onto the first column filled with TEVA resin (Triskem Int.). On reaching the transient equilibrium, the daughter 226 Th was separated and eluted with strong HCl solution. The role of the second column was to reduce quickly the acidity of 226 Th solution, i.e., a sorbent for second column was to retain 226 Th from the strong HCl solution and to desorb it into a diluted solution. Three extraction chromatographic resins eligible for this purpose were considered: TRU resin, UTEVA resin, and DGA resin (all Triskem Int.). According to the reported data [37][38][39] obtained in static conditions and shown in Figure 3a, the resins can be arranged in a row with respect to Th(IV) retention from <5 M HCl solution: In order to evaluate the feasibility of a two-column 230 U/ 226 Th generator, column experiments on 226 Th sorption from 7 M HCl and its desorption with diluted HCl solutions were carried out.

Elution of 226 Th from the Second Column with HCl Solutions
First, the transfer of 226 Th to a second column was investigated ( Figure 4a, Step 1). 226 Th was easily stripped off the parent column with 7 M HCl solution, the concentration corresponding to the maximum of U(VI) sorption on TEVA resin ( Figure 2). The integral 226 Th elution curve (blue line in Figure 5a) indicates that the solution volume of 1.5 mL was sufficient to wash out ≥99% of 226 Th. DGA resin and TRU resin display high adsorption of 226 Th from 7 M HCl solution (Figure 3a), the values of k′ Th(IV) attain 10 4 . When the second column was filled with 0.1 mL of these resins and connected directly to the exit of the parent column, 226 Th was completely adsorbed onto the second column. In contrast, the values of k′ Th(IV) on UTEVA resin are below 10 under the same conditions. Therefore, the quantity of UTEVA resin in the second column was increased up to 1 mL to ensure a tolerable breakthrough of 226 Th of less than 3% (red line in Figure 5a). In order to evaluate the feasibility of a two-column 230 U/ 226 Th generator, column experiments on 226 Th sorption from 7 M HCl and its desorption with diluted HCl solutions were carried out.

Elution of 226 Th from the Second Column with HCl Solutions
First, the transfer of 226 Th to a second column was investigated ( Figure 4a, Step 1). 226 Th was easily stripped off the parent column with 7 M HCl solution, the concentration corresponding to the maximum of U(VI) sorption on TEVA resin ( Figure 2). The integral 226 Th elution curve (blue line in Figure 5a) indicates that the solution volume of 1.5 mL was sufficient to wash out ≥99% of 226 Th. DGA resin and TRU resin display high adsorption of 226 Th from 7 M HCl solution (Figure 3a), the values of k Th(IV) attain 10 4 . When the second column was filled with 0.1 mL of these resins and connected directly to the exit of the parent column, 226 Th was completely adsorbed onto the second column. In contrast, the values of k Th(IV) on UTEVA resin are below 10 under the same conditions. Therefore, the quantity of UTEVA resin in the second column was increased up to 1 mL to ensure a tolerable breakthrough of 226 Th of less than 3% (red line in Figure 5a). Molecules 2023, 28, x FOR PEER REVIEW 5 of 16    The transferred 226 Th was eluted with different HCl solutions as shown in Figure  (Step 2). The DGA resin exhibits the greatest retention of 226 Th among the studied res from diluted hydrochloric solutions (Figure 3a). Our results of column experiments w in a good agreement with the k' data. The elution of 226 Th with 0.3 M HCl solution, wh is the most favorable for 226 Th desorption, resulted in 40% of 226 Th yield in 6 mL of elu For other HCl concentrations, the 226 Th yield was even lower.
The efficiency of 226 Th desorption from the second columns filled with TRU a UTEVA resins versus the concentration of hydrochloric solution is presented in Figure  The optimal HCl concentration range of 226 Th desorption was 0.4-1 M for TRU resin a 0.5-2 M for UTEVA resin. Typical 226 Th elution curves ( Figure 6) display that 226 Th w  The efficiency of 226 Th desorption from the second columns filled with TRU and UTEVA resins versus the concentration of hydrochloric solution is presented in Figure 5b. The optimal HCl concentration range of 226 Th desorption was 0.4-1 M for TRU resin and 0.5-2 M for UTEVA resin. Typical 226 Th elution curves ( Figure 6) display that 226 Th was completely eluted in~1 mL of eluate. It is interesting to note that the width of 226 Th chromatographic peaks from the TRU resin and UTEVA resin columns was almost the same, although the bed volume of UTEVA resin was 10 times larger than the TRU resin.
cules 2023, 28, x FOR PEER REVIEW 6 of completely eluted in ~1 mL of eluate. It is interesting to note that the width of 226 Th ch matographic peaks from the TRU resin and UTEVA resin columns was almost the sam although the bed volume of UTEVA resin was 10 times larger than the TRU resin.
(a) (b) Despite the fact that the HCl concentration of the solution entering the second c umn for 226 Th desorption (Step 2) was relatively low, the eluate acidity from both TR resin and UTEVA resin columns was 3-4 M [H + ]. The detailed titration curves of elua collected by portions ( Figure 6) demonstrate that 226 Th is eluted on the drastic HCl co centration gradient when one solution is replaced by another. The maximum of 226 Th ch matographic peak corresponds to H + concentration around 4 M. Therefore, the use of lute HCl solutions allowed us to decrease eluate acidity by only a factor of two.

Elution of 226 Th from the Second Column with Citric Buffer Solutions
After transferring 226 Th from the parent column to a second one containing TR UTEVA or DGA resin, 226 Th can be desorbed with a neutral citric buffer solution. The e ciency of 226 Th desorption was studied as a function of H3Cit (pH 5.0) concentration in t range of 10 −4 M-10 −1 M. The dependencies plotted on the graph (Figure 7) are arranged the order reflecting the above-mentioned sequence of resin affinity for thorium (IV). F the studied resins, 0.1 M citric buffer solution fully recovered 226 Th. Despite the fact that the HCl concentration of the solution entering the second column for 226 Th desorption (Step 2) was relatively low, the eluate acidity from both TRU resin and UTEVA resin columns was 3-4 M [H + ]. The detailed titration curves of eluate collected by portions ( Figure 6) demonstrate that 226 Th is eluted on the drastic HCl concentration gradient when one solution is replaced by another. The maximum of 226 Th chromatographic peak corresponds to H + concentration around 4 M. Therefore, the use of dilute HCl solutions allowed us to decrease eluate acidity by only a factor of two.

Elution of 226 Th from the Second Column with Citric Buffer Solutions
After transferring 226 Th from the parent column to a second one containing TRU, UTEVA or DGA resin, 226 Th can be desorbed with a neutral citric buffer solution. The efficiency of 226 Th desorption was studied as a function of H 3 Cit (pH 5.0) concentration in the range of 10 −4 M-10 −1 M. The dependencies plotted on the graph (Figure 7) are arranged in the order reflecting the above-mentioned sequence of resin affinity for thorium (IV). For the studied resins, 0.1 M citric buffer solution fully recovered 226 Th.
Typical curves of 226 Th elution from the UTEVA resin and DGA resin columns consisted of the only chromatographic peak within the given range of citric acid concentration (Figure 8a). Similar to the 226 Th elution with dilute hydrochloric solutions, the peak maximum followed the acidity gradient. Otherwise, two chromatographic peaks were observed when 226 Th was eluted from the TRU column with citric buffer solutions (Figure 8b). The first peak was in the same way related to the HCl concentration gradient. The position of maximum V max of the second peak, as well as the capacity factor k defined as k = V max −V c V c [37] (V c is free volume of sorbent in a column), depended on the citric acid concentration.
After transferring 226 Th from the parent column to a second one containing TRU, UTEVA or DGA resin, 226 Th can be desorbed with a neutral citric buffer solution. The efficiency of 226 Th desorption was studied as a function of H3Cit (pH 5.0) concentration in the range of 10 −4 M-10 −1 M. The dependencies plotted on the graph (Figure 7) are arranged in the order reflecting the above-mentioned sequence of resin affinity for thorium (IV). For the studied resins, 0.1 M citric buffer solution fully recovered 226 Th.  Typical curves of 226 Th elution from the UTEVA resin and DGA resin columns consisted of the only chromatographic peak within the given range of citric acid concentration (Figure 8a). Similar to the 226 Th elution with dilute hydrochloric solutions, the peak maximum followed the acidity gradient. Otherwise, two chromatographic peaks were observed when 226 Th was eluted from the TRU column with citric buffer solutions ( Figure  8b). The first peak was in the same way related to the HCl concentration gradient. The position of maximum Vmax of the second peak, as well as the capacity factor k′ defined as According to the literature data, various complexes of Th(IV) are coexisting in citric acid media depending on pH values and salinity [40,41]. At pH 5-6, the predominant species are ThCit3 5− and ThCit2(OH)2 4− [40] with cumulative formation constants (logβ) of 28 and 15, respectively. In more acidic solution (pH < 1), the speciation shifts toward cation forms of Th(IV), e.g., Th 4+ and ThCit + (logβ = 14). It is evident that all these species may exhibit different affinities to the resins and the detailed analysis is complicated. However, in the case of TRU resin, the dependence of k′ Th(IV) on citric acid concentration may be expressed by a simple correlation helpful for practice use:

= 1 + • H Cit
where and are empirical constants. Satisfactory values of 226 Th yield (>90%) in a small amount of eluate (1-1.5 mL) were obtained for all the studied resins, the eluate characteristics are listed in Table 1.  According to the literature data, various complexes of Th(IV) are coexisting in citric acid media depending on pH values and salinity [40,41]. At pH 5-6, the predominant species are ThCit 3 5− and ThCit 2 (OH) 2 4− [40] with cumulative formation constants (logβ) of 28 and 15, respectively. In more acidic solution (pH < 1), the speciation shifts toward cation forms of Th(IV), e.g., Th 4+ and ThCit + (logβ = 14). It is evident that all these species may exhibit different affinities to the resins and the detailed analysis is complicated. However, in the case of TRU resin, the dependence of k Th(IV) on citric acid concentration may be expressed by a simple correlation helpful for practice use: where k 0 and a are empirical constants.
Satisfactory values of 226 Th yield (>90%) in a small amount of eluate (1-1.5 mL) were obtained for all the studied resins, the eluate characteristics are listed in Table 1. It was found that eluate acidity remained relatively excessive for immediate synthesis of labeled compounds. In order to maintain 226 Th on the second column during the substitution of the acidic medium with the neutral one, the influence of nitrate ions was studied.

Stabilization of 226 Th on the Second Column before Elution
The ability of Th(IV) to form stable anionic complexes with nitrate ions is widely used to separate it from other elements. Comparison of k values for DGA, TRU, and UTEVA resins reveals higher sorption of Th(IV) from nitric solutions (Figure 3b) than from hydrochloric ones (Figure 3a), especially for the acidity below 1 M. Taking this fact into account, we modified the procedure of 226 Th production from the two-column 230 U/ 226 Th generator.
The initial part of modification consisted of the pre-treatment of the second column. Before transferring 226 Th from the parent column (Step 1), 10 mL of HNO 3 solution of a certain concentration was passed through the second column at a flowrate of 1 mL/min. For the DGA and TRU resins, the HNO 3 concentration was 0.1 M, which corresponds to moderate values of the capacity factor 150 < k Th(IV) < 350 (Figure 3b). In the case of UTEVA resin, the values of k Th(IV) for dilute nitric acid solutions are small; they grow with an increasing acid concentration and reach values around 100 in the region of 3-4 M HNO 3 .
Starting from these data obtained in static conditions, the UTEVA column was pretreated with a HNO 3 solution of various concentrations and 226 Th losses during Step 1 were studied. The results presented in Figure 9 display that the 226 Th losses when transferring from the TEVA column to UTEVA one with 7 M HCl solution noticeably diminished along with increasing the concentration of HNO 3 used for UTEVA pre-treatment. For 3 M HNO 3 solution, the breakthrough of 226 Th began after passing not less than 2.5 mL of 7 M HCl.
Molecules 2023, 28, x FOR PEER REVIEW 8 of 16 It was found that eluate acidity remained relatively excessive for immediate synthesis of labeled compounds. In order to maintain 226 Th on the second column during the substitution of the acidic medium with the neutral one, the influence of nitrate ions was studied.

Stabilization of 226 Th on the Second Column before Elution
The ability of Th(IV) to form stable anionic complexes with nitrate ions is widely used to separate it from other elements. Comparison of k' values for DGA, TRU, and UTEVA resins reveals higher sorption of Th(IV) from nitric solutions (Figure 3b) than from hydrochloric ones (Figure 3a), especially for the acidity below 1 M. Taking this fact into account, we modified the procedure of 226 Th production from the two-column 230 U/ 226 Th generator.
The initial part of modification consisted of the pre-treatment of the second column. Before transferring 226 Th from the parent column (Step 1), 10 mL of HNO3 solution of a certain concentration was passed through the second column at a flowrate of 1 mL/min. For the DGA and TRU resins, the HNO3 concentration was 0.1 M, which corresponds to moderate values of the capacity factor 150 < k' Th(IV) < 350 (Figure 3b). In the case of UTEVA resin, the values of k' Th(IV) for dilute nitric acid solutions are small; they grow with an increasing acid concentration and reach values around 100 in the region of 3-4 M HNO3.
Starting from these data obtained in static conditions, the UTEVA column was pretreated with a HNO3 solution of various concentrations and 226 Th losses during Step 1 were studied. The results presented in Figure 9 display that the 226 Th losses when transferring from the TEVA column to UTEVA one with 7 M HCl solution noticeably diminished along with increasing the concentration of HNO3 used for UTEVA pre-treatment. For 3 M HNO3 solution, the breakthrough of 226 Th began after passing not less than 2.5 mL of 7 M HCl. In addition, a variation of Step 1 was tested (Figure 4b) that provided the flow of 7 M HCl solution carrying 226 Th to be equally mixed with the flow of 3 M HNO3 solution at the entrance of the UTEVA column ("two flows" variation). As a result, 226 Th breakthrough In addition, a variation of Step 1 was tested (Figure 4b) that provided the flow of 7 M HCl solution carrying 226 Th to be equally mixed with the flow of 3 M HNO 3 solution at the entrance of the UTEVA column ("two flows" variation). As a result, 226 Th breakthrough was not observed even after passing 20 mL of 7 M HCl (>20 bed volumes).
The other part of the second column modification was carried out after Step 1 and involved substituting the 7 M HCl medium with dilute or neutral one. Solutions of 0.1 M HNO 3 , 0.15 M NaCl, and 0.15 M NaNO 3 were studied. Full change in medium in the DGA column took place after passing 1.0-1.2 mL of each solution, and 226 Th losses did not exceed 3%. In the case of TRU column, 226 Th was partially washed out on the HCl concentration gradient (similar to Figure 6a) regardless of the substituting solution, its losses ranged from 10% to 40% and were poorly reproducible. The UTEVA resin retained 226 Th well when the solutions of 0.1 M HNO 3 and 0.15 M NaNO 3 were passed through the second column, whereas the 0.15 M NaCl solution washed out up to 28% of 226 Th ( Figure 10).
Molecules 2023, 28, x FOR PEER REVIEW column took place after passing 1.0-1.2 mL of each solution, and 226 Th losses did ceed 3%. In the case of TRU column, 226 Th was partially washed out on the HCl tration gradient (similar to Figure 6a) regardless of the substituting solution, it ranged from 10% to 40% and were poorly reproducible. The UTEVA resin retain well when the solutions of 0.1 M HNO3 and 0.15 M NaNO3 were passed through ond column, whereas the 0.15 M NaCl solution washed out up to 28% of 226 Th (Fig   Figure 10. 226 Th losses during the substitution of the 7 M HCl medium on the UTEVA resin with neutral salt solutions. Having replaced the strongly acidic medium in the second column, 226 Th was out with a citric buffer solution (pH 5.0) of various concentrations (Figure 11a). Th obtained for the UTEVA column: • without the pre-treatment and medium substitution (green line in Figure 7); • with the pre-treatment and substitution of 7 M HCl with 0.15 M NaNO3 soluti solid line in Figure 11a); • with the pre-treatment, "two flows" variation and the substitution (blue das in Figure 11a) allow us to suggest that increasing contact time of the UTEVA resin and nitric solu to an increasing difficult 226 Th recovery. The relative positions of 226 Th elution curv ure 11b) were also in line with these suggestions. Having replaced the strongly acidic medium in the second column, 226 Th was washed out with a citric buffer solution (pH 5.0) of various concentrations (Figure 11a). The results obtained for the UTEVA column: • without the pre-treatment and medium substitution (green line in Figure 7); • with the pre-treatment and substitution of 7 M HCl with 0.15 M NaNO 3 solution (blue solid line in Figure 11a); • with the pre-treatment, "two flows" variation and the substitution (blue dashed line in Figure 11a) allow us to suggest that increasing contact time of the UTEVA resin and nitric solution led to an increasing difficult 226 Th recovery. The relative positions of 226 Th elution curves (Figure 11b) were also in line with these suggestions.
Moreover, the influence of the contact time was evaluated in parallel experiments that included the 3 M HNO 3 pre-treatment of the UTEVA column, the HCl substitution with 0.15 M NaNO 3 solution, and the 226 Th elution with a 10 −3 M citric buffer solution. As the pre-treatment duration increased from 10 min to 10 h, the efficiency of 226 Th production decreased from 70% (see in Figure 11a) to zero. The behavior of the UTEVA resin may be explained by the fact that the k Th(IV) dependence in nitric solutions is higher than the hydrochloric solutions over the entire concentration range (Figure 3). solid line in Figure 11a); • with the pre-treatment, "two flows" variation and the substitution (blue dashed line in Figure 11a) allow us to suggest that increasing contact time of the UTEVA resin and nitric solution led to an increasing difficult 226 Th recovery. The relative positions of 226 Th elution curves (Figure 11b) were also in line with these suggestions. Two most effective procedures for obtaining 226 Th from the two-column 230 U/ 226 Th generator are presented in Table 2. The overall time of 226 Th eluate production was within 5-7 min. The time expenditure was slightly shorter when the DGA column was used, since its size was 10 times smaller than the UTEVA column. Meanwhile, the use of UTEVA resin for the second column resulted in high yield of 226 Th in a less concentrated solution (0.01-0.05 M H 3 Cit, pH 4.5-5.0).

Long-Term Performance of the Two-Column 230 U/ 226 Th Generator
Following the developed procedure, the solution of 7 M HCl was only passed through the parent column containing 230 U adsorbed on TEVA resin. The distribution of 230 U along with the length of parent column was monitored throughout the generator lifetime ( Figure A2). Usually, two cycles of 226 Th production per weekday were performed over 2 months. The total volume of 7 M HCl solution that passed through the parent column was about 200 mL including accessory operations and a loading of a second portion of 230 U. According to the calculations illustrated by Figure A3, one third of the initial amount of 230 U was additionally accumulated from 230 Pa in 27-28 days after the first separation, and then loaded onto the parent column.
Due to the two-column scheme of 226 Th production, the proposed generator provided deep purification 226 Th from 230 U. It was found that the 230 U impurity in the 226 Th eluates did not exceed 0.01%, which was at least one order of magnitude better in comparison with the reported literature data [13].
In this work, we used low 230 U activities that do not lead to radiolysis and destruction of the sorbent. Influence of these processes on the generator performance is to be investigated in future studies.
Citric buffer solutions (10 −4 -10 −1 M, pH 5.0 ± 0.1) were prepared by dissolving the corresponding solid acid sample and adding small portions of 1 M NaOH to obtain a solution with the required pH value.
Measurements of operational pH values were performed with an Orion 2 Star Benchtop pH meter using an Orion 8103SC combination pH electrode. Commercial pH Titrisol buffer concentrates (Merck p.a.) were used to calibrate the setup at room temperature.
Acid-base titration with indicators methyl orange and phenolphthalein were used to determine the acid content in commercial solutions of concentrated HCl and HNO 3 , as well as in the 226 Th containing eluate.
The experiments were carried out at a temperature of 21 ± 2 • C.

Gamma-ray Spectroscopy
The measurement of radionuclide activities was performed by γ-ray spectrometry using a high resolution HP Ge detector (ORTEC GEM15P4-70). Samples were counted at different detector-source distances respecting a level of dead-time of less than 10%. The detector efficiency at the used distances were determined with standard calibration sources. Net peak areas in the detected photopeaks were evaluated by means of the GammaVision32 software.
The characteristic γ-ray emission of 226 Th (111.1 keV, 3.29%) and 222 Ra (324.3 keV, 2.77%) [2] were used for 230 U and 226 Th activity quantification of various generator testing samples respecting the transient equilibrium of daughter radionuclides.

Target Preparation and Irradiation, and 230 U Isolation
Metallic thorium supplied by Institute for Physics and Power Engineering (IPPE, Russia) was used as target material. Thorium plates of (2.2 × 2.5) cm 2 approximate dimensions with thickness of 1.5-2.0 mm were fabricated and packed in copper and aluminum foil envelopes, which served for beam monitoring purposes, as well. Each package was encapsulated in a graphite shell sealed with high-temperature silicone adhesive. Several targets were irradiated at the linear proton accelerator of the Institute for Nuclear Research of the Russian Academy of Sciences (INR RAS, Moscow, Russia) [42] with an initial energy of 120-130 MeV. The beam current and total beam charge were 3-5 µA and 12-18 µA·h, respectively.
The dissolution of the irradiated thorium was performed as described previously [18,43] 4 to 5 days after the end of bombardment (EOB). The protactinium fraction including 230 Pa was recovered from the solution according to the procedure reported in [15] and remained in 7 M HCl/0.1 M HF solution for 230 U accumulation during 27-28 days. Traces of Nb (mainly 95 Nb) and Ru ( 103,106 Ru) radionuclides were impurities of 230 Pa/ 230 U.
A chromatographic technique close to the one developed by A.W. Knight [44] was implemented for separation of 230 U from 230 Pa. The solution with radionuclides was loaded onto a column filled with 2 mL of TEVA resin. Due to the presence of fluoride ions, Pa(V) together with Ru(IV) were eluted, while U(VI) and Nb(V) were retained on the resin. The column was washed with 7 M HCl/0.1 M HF solution to remove Pa(V). The washing was added to the Pa(V) eluate and the combined solution was maintained for the next 230 U accumulation. Then, having the column washed with 7 M HCl solution, U(VI) and a part of Nb(V) were desorbed with 0.1 M HCl solution. The desorbate was adjusted to 3 M HCl by adding concentrated hydrochloric acid, and following the known procedure [13,45], this solution was passed through a column filled with 1 mL of DGA resin. The uranium fraction was adsorbed, while the 95 Nb was washed out of the column. Finally, 230 U was eluted with a small amount of 0.1 M HCl solution.

Preparation of a Parent 230 U Column
A plastic column of~5 mm in diameter was filled with 1 mL of TEVA resin equilibrated with 7 M HCl. The resin was fixed inside by two frits at the bottom and at the top of column. The 230 U solution was evaporated and reconstituted in 7 M HCl. The resulting solution was passed through the column followed by washing with a solution of 7 M HCl. The uranium was adsorbed under these conditions, the loaded activity of 230 U was 300-350 kBq. Approximately a month after the 230 U isolation and loading, a second part of 230 U was accumulated, separated from 230 Pa as described above and added to the column.
The prepared column served as parent one, and it could work as a one-column generator providing 226 Th elution in 7 M HCl. A typical differential curve of 226 Th elution is shown in Figure A1. Furthermore, the parent column was a part of the two-column generator schemes.

Two-Column 230 U/ 226 Th Generator Scheme and 226 Th Elution Cycle
A general scheme of the 230 U/ 226 Th generator comprised two columns connected in series via a three-valve cock as shown in Figure 4. The parent TEVA column containing 230 U was the first column and a column filled with TRU, UTEVA or DGA resin served as a second one that could be interchanged when necessary. A milking procedure included two common steps: (1) Transfer 226 Th with 7 M HCl solution from the parent column to the second one (Figure 4a,b); (2) 226 Th elution with diluted HCl or citric buffer solution (Figure 4c). Eluates were collected for measurement of 226 Th and 230 U activity and eluate acidity. Flow rates of solutions passing through the columns were maintained and controlled with a peristaltic pump. The values of flow rate were 1 and 0.6 mL/min for Steps 1 and 2, respectively.
The TRU or DGA resin was loaded into a plastic column of~3 mm in diameter, the height of resin bed was 11-12 mm (resin volume~0.1 mL). Diameter of the column for the UTEVA resin was~5 mm, the height of the UTEVA bed was 55-58 mm (~1 mL). Each column was equipped with bottom and top frits.
For some experiments with the UTEVA column, we modified Step 1 as it is shown in Figure 4b. A flow of 7 M HCl solution (0.5 mL/min) after passing the parent 230 U column was mixed with a flow of 3 M HNO 3 solution (0.5 mL/min) resulting in 1 mL/min flow of 3.5 M HCl/1.5 M HNO 3 solution at the entrance of the UTEVA resin column.

230 U Measurements
The activity of 230 U in eluates was usually measured overnight for complete 226 Th decay. 230 U was assayed by γ-ray spectroscopy via the daughter radionuclides 226 Th and 226 Ra.
Distribution of 230 U along the length of TEVA resin column was monitored by successive scanning of the column through a 4 mm wide slit between lead blocks. The measure-ments were performed after 230 U loading onto the TEVA resin column and regularly after milking.

Conclusions
We have proposed and tested the proof-of-concept of a two-column 230 U/ 226 Th generator for rapidly producing 226 Th amenable to further labeling. The first 230 U column with TEVA resin furnished 226 Th in 7 M HCl solution. The second column retained 226 Th from the strongly acidic solution, and then released it with a diluted hydrochloric or neutral citric buffer solution. Analysis based on the dependence of the capacity factor k Th(IV) on the concentration of hydrochloric and nitric acid allowed us to consider DGA, TRU, and UTEVA resins as promising sorbents for the second column.
High yields (>97%) of 226 Th elution from TRU and UTEVA resins with a small volume (~1 mL) of diluted HCl solutions were obtained. However, the resulting acidity of the eluate was 3-4 M [H + ] regardless of the solution concentration entering the second column. The titration analysis displayed that 226 Th was eluted on the HCl concentration gradient when one solution was replaced by another.
Elution of 226 Th transferred to the second column containing DGA, TRU or UTEVA resin was studied with citric buffer solutions (pH 5.0) in two modes. Direct 226 Th desorption was also influenced by the acidity gradient. While 226 Th was stripped off the UTEVA and DGA columns in one chromatographic peak, a typical curve of 226 Th elution from the TRU column consisted of two chromatographic peaks within the studied range of citric acid concentration. The first peak followed the HCl concentration gradient, and the second one may be attributed to 226 Th complexation with citrate ions in the course of TRU column elution. Satisfactory yields were achieved by 226 Th elution from the second column filled with any of the studied resins. For TRU and DGA resins, 1-1.5 mL of 0.1 M H 3 Cit (pH 5.0) solution extracted more than 90% of 226 Th, while the UTEVA resin column demonstrated similar effectiveness with less concentrated citric buffer solutions (down to 10 −3 M H 3 Cit). The acidity of citric eluates was about two times lower than the diluted HCl solution's eluates but still relatively high for immediate labeling.
Neutral citric-buffered 226 Th eluates were obtained when nitrate ions were introduced. The second column was initially put in contact with a nitric acid solution. Then, after 226 Th transfer from the parent column, the acidic medium of the second column was substituted with the neutral one maintaining 226 Th immobile. Solutions of 0.15 M NaCl and of 0.15 M NaNO 3 were used for the DGA and UTEVA column, respectively. Finally, 226 Th was extracted with citric buffer solution: 0.1 M H 3 Cit from the DGA column and 0.01-0.05 M H 3 Cit from the UTEVA one. Therefore, one cycle of generator milking took 5-7 min and produced >90% of 226 Th in 1.5 mL of eluate, pH 4.5-5.0.
The proposed two-column 230 U/ 226 Th generator was tested over 2 months including a second loading of 230 U additionally accumulated from 230 Pa. The 230 U impurity in the 226 Th eluate was less than 0.01% allowing its use directly in synthesis of radiopharmaceutical compounds.        [15]).